Pancreatic lipase analysis, which is useful for pancreatic disease diagnosis, is often carried out by measuring pancreatic lipase under conditions in which a micellar substrate is dispersed in water in view of the fact that pancreatic lipase functions in oil-water interface. This is because non-pancreatic lipase such as lipoprotein lipase, liver lipase, or esterase reacts with a substrate solubilized with a surfactant or the like or with glyceride of fatty acid having short alkyl chains. Thus, it is considered that a technically important point for development of dry analytical elements for pancreatic lipase is the incorporation of glyceride of a long-fatty-acid (serving as a substrate), which is specific to pancreatic lipase, into such analytical element, in a state that the glyceride is specific to pancreatic lipase.
Dry analytical elements used for lipase analysis are roughly classified into two groups. An example of a first group is a multilayer dry analytical element (JP Patent Publication (Kokai) No. 59-48098 A (1984)) according to a method wherein triglyceride is used as a substrate and is converted into a dye via glycerine and hydrogen peroxide. According to the first disclosed method, a triglyceride having a long-chain alkyl group having at least 8 carbon atoms at an ester position (α position) and having a short-chain alkyl group at two other esters each is used as a substrate, 1,2 diacetylglyceride generated by lipase in a specimen is converted into glycerine with the use of an esterase (namely, acetinase), and glycerine is converted into a dye. The above method is a convenient and highly accurate lipase measurement method. However, it has been reported that selectivity with respect to pancreatic lipase is not high, and thus the method is problematic for diagnosis of pancreatic diseases (Clin. Chem., 37/3, 447-451 (1991)).
Next, a method using a dry chemistry reagent for pancreatic lipase analysis, such reagent comprising triglyceride (serving as a substrate) comprising a long chain fatty acid (e.g., triolein) having 14 to 20 carbon atoms, monoglyceride lipase, and a glycerine measurement reagent is disclosed in JP Patent Publication (Kokai) No. 4-316500 A (1992). Further, a method wherein a highly accurate multilayer analytical element is prepared according to the above method and fine particles are further incorporated into the element in order to improve lipase reactivity is disclosed in JP Patent Publication (Kokai) No. 2002-125699 A. In the method of JP Patent Publication (Kokai) No. 4-316500 A (1992), highly fat-soluble substrate is incorporated, so that a protective colloid such as gum Arabic is used for aqueous system dispersion using ultrasonic dispersion (JP Patent Publication (Kokai) No. 4-316500 A (1992): Examples). Accordingly, it is necessary to maintain the reproducibility of substrate dispersion and uniformity in particle size distribution, and it is thought that production of such element is difficult.
For instance, JP Patent Publication (Kokai) No. 4-316500 A (1992) contains the following description: “triglyceride, such as triolein, comprising a long chain fatty acid in each of three ester positions has the property of being emulsified with difficulty. Thus, even if a solution in which triolein has been uniformly emulsified and dispersed via agitation or by physical shearing force generated by ultrasound waves or the like is added in the presence of a surfactant or a protective colloid upon preparation of a dry reagent, water serving as a dispersion medium disappears when the reagent becomes dry, and thus an emulsified product aggregates or coalesces so as to adhere to the surface of a development layer, resulting in significant reduction in the surface area in oil-water interface. Upon measurement, even if a specimen (liquid) containing lipase is allowed to react with such dry reagent, triolein remains in a state of aggregating or coalescing and thus does not return to the original state of being dispersed because of lack of physical shearing force. The reaction field of lipase is an oil-water interface. Thus, a decrease in the surface area of an oil-water interface is thought to cause a decrease in reaction rate.”
An example of a second group is a dry analytical element obtained by a method using 1,2-O-dilauryl-rac-glycero-3-glutaric acid/resorufin ester serving as a dye-releasing substrate (JP Patent Publication (Kokai) No. 9-154598 A (1997)). Such method is a preferable because high specificity with respect to pancreatic lipase can be achieved and a glycerine coloring system is unnecessary. However, the substrate incorporated into a dry analytical element is highly likely to degrade. Thus, such dry analytical element has still not been available in practice, although it has been attempted to separate a low-pH layer containing a lipase substrate from another high-pH reagent layer. In addition, in such case, an ether system solvent that is thought to be preferable for dissolution of a substrate imposes significant environmental burdens and thus is seriously problematic in terms of production suitability in the present situation in which environmentally-friendly designs are strongly required. Further, the relatively high price of such a substrate is also problematic in terms of practical use.
As described above, the product disclosed in JP Patent Publication (Kokai) No. 59-48098 A (1984) is still the only commercially available dry analytical element for lipase analysis, although the product has low pancreatic lipase specificity. Thus, dry analytical elements that are excellent in terms of reliability for diagnosis of pancreatic diseases have been desired in the market.